PKR, the RNA-dependent protein kinase, is a member of the elF2 alpha kinase family that controls the rate of translation initiation in human cells in response to various environmental stresses. PKR has been implicated in the interferon antiviral response, tumor suppression, differentiation and apoptosis. However, it has not been established to what extent all these activities involve RNA-induced phosphorylation of elF2 alpha. Double stranded RNA (dsRNA) activates PKR in a manner that involves binding to both its double stranded RNA-binding motifs (dsRBMs), conformational changes in the kinase domain and dimerization, although the molecular details of this process remain to be elucidated. Proteins that activate PKR, as well as both RNA and proteins that block PKR activation have also been identified. In these cases, the mechanism of kinase regulation is often poorly defined. No high-resolution structures of full-length PKR or any PKR.RNA complexes have been reported to date. Furthermore, although PKR shows selectivity in its binding to dsRNA, the basis for this binding selectivity and its importance in kinase regulation is not known. The research proposed here is designed to further our understanding of RNA binding and regulation of PKR's kinase activity and to aid in defining PKR's role in signal transduction pathways. This will be accomplished by using both biophysical and chemical approaches to identify important contacts in PKR.RNA complexes. New reagents useful for probing the function of PKR on cell signaling pathways will be developed including a PKR mutant and ATP analogs to be used for the identification of PKR substrates found in cell lysates. Small molecule libraries of potential inhibitors of PKR will be prepared and screened. RNA aptamers that bind PKR's dsRBMI in a ligand-regulated fashion will be evolved in vitro, providing a method to control PKR activity temporally. In addition, experiments to further elucidate the mechanism by which RNA regulates PKR's kinase activity will be performed. These will include studies designed to the correlate the relative positions of binding sites for PKR's two dsRBMs on an RNA molecule with that RNA's efficacy as a kinase activator.